Negative strand RNA viruses are the cause of numerous diseases in humans and animals. Detection and quantification of negative strand RNA viruses in biological specimens are thus of importance to both clinicians and researchers.
Negative strand RNA viruses can comprise segmented RNA or non-segmented RNA. In a non-segmented negative strand RNA virus, the entire viral genome is comprised by a single negative-sense RNA molecule, whereas in a segmented negative stand RNA virus, the viral genome is comprised by a plurality of negative-sense RNA segments. Among the segmented negative strand RNA viruses are the Arenaviridae, the Bunyaviridae and the Orthomyxoviridae, comprising two, three and six to eight segments, respectively. Each segment of vRNA comprises, in 5′ to 3′ order, a 5′ untranslated region (“UTR”), at least one anti-sense open reading frame sequence encoding a viral polypeptide, and a 3′ UTR. The UTRs are recognized by and/or bind to at least one protein involved with segmented negative strand RNA replication and/or gene expression, such as an RNA-dependent RNA polymerase. It is now generally accepted that expression of a protein encoded by a segment requires that the segment have precisely defined 5′ and 3′ terminal sequences.
U.S. Pat. Nos. 5,591,579 and 5,851,757, to Olivo et al. disclose cell lines and methods for detecting the presence of non-segmented RNA viruses in biological specimens. The cell lines are transformed with a DNA molecule that comprises a promoter capable of recognition by a cellular DNA-dependent RNA polymerase. The promoter, which is capable of directing the transcription of a cDNA of a structurally defective RNA virus, is linked to a reporter cDNA. The RNA molecules transcribed by the DNA-dependent RNA polymerase are not capable of causing the translation of reporter cDNA unless an active related virus is provided that contributes certain trans-acting viral enzymes.
U.S. Pat. No. 6,270,958 to Olivo et al. discloses a diagnostic assay for detecting a non-segmented negative-strand RNA virus in a biological specimen. In the disclosed assay, a genetically engineered cell expresses a minigenome or miniantigenome of a negative strand RNA virus. The minigenome or miniantigenome comprises a reporter gene or its complement, and flanking sequences from viral RNA. The cell also comprises recombinant DNA sequences each comprising a promoter directing the expression of nucleocapsid protein of the virus. Expression of the reporter gene in these cell lines depends upon the presence of the nucleocapsid proteins as well as infection of the cell with the virus.
Because expression of a polypeptide encoded by a segment of a segmented negative strand RNA virus requires precisely defined 3′ and 5′ terminal sequences, investigators have exploited the biochemical properties of RNA polymerase I (Pol I) to investigate expression of cDNAs of segments of segmented negative strand RNA viruses. Pol I is believed to initiate and terminate transcription at precise nucleotide sequences. In this regard, Zobel et al., Nucleic Acids Research 21: 3607-3614, 1993, and Neumann et al., Virology 202: 477-479, 1994 generated recombinant DNA constructs comprising a promoter recognized by Pol I (a “Pol I promoter”) derived from a mouse ribosomal RNA (rDNA) gene. The Pol I promoter in these recombinant DNA constructs directed transcription (presumably by Pol I) of an operably linked DNA sequence comprising a Pol I transcription start site, an influenza virus segment comprising a 5′ untranslated region (UTR) of an influenza virus RNA segment, a sequence complementary to a chloramphenicol acetyltransferase (CAT) coding sequence (replacing a hemagglutinin cDNA), and a 3′ UTR, as well as a transcription terminator sequence recognized by Pol I polymerase (a “Pol I terminator”). Cells infected with influenza virus that were transfected with this DNA construct expressed CAT activity, suggesting that a virus-encoded RNA-dependent RNA polymerase was able to use the Pol I transcript as a template, resulting in a translatable mRNA encoding an active CAT enzyme.
These and similar studies have led to complete production of influenza virus using recombinant DNAs comprising cDNAs of influenza virus segments. In this connection, Neumann et al., Proceedings of the National Academy of Sciences USA 96: 9345-9350, 1999, and Hoffmann et al., Proceedings of the National Academy of Sciences USA 97: 6108-6113, 2000 disclose “reverse genetics” DNA transfection systems for generation of influenza A viruses entirely from cloned cDNAs. In these studies, cells were transfected with a first series of plasmids, each plasmid comprising a DNA sequence comprising a Pol I promoter operably linked to a cDNA of a vRNA segment, as well as a second series of plasmids, each plasmid comprising a Pol II promoter operably linked to a cDNA encoding a viral protein. The viral proteins transcribed in this system included viral nucleoprotein (NP) and polymerase complex proteins (comprising proteins PA, PB1, and PB2).